Misincorporated nucleotides

Mutagenic PGR. More recently, methods have been developed to use the PGR reaction to randomly mutagenize a defined sequence (25). The Taq polymerase used in PGR misincorporates nucleotides in a random fashion if manganese dichloride [7773-01 -5] MnGl2, is included in the reaction buffer during PGR. The Hbrary of mutagenized PGR products can be screened for the desired phenotype. 

For most applications the error rate of the polymerase has no effect on the quality of the sequence information obtained. This is because the amplified product (sequencing template) is a population of molecules with only a relatively small fraction of the copies containing misincorporated nucleotides at any one position (Table II). The fraction of template molecules that will contain a misincorporated base is dependent on the starting number of target molecules and the cycle in which the error occurred. The fraction is very small, undetectable in most applications, if the number of starting molecules is at least 10. 

The 3 —>5 exonuclease activity of DNA polymerase I, at least, functions to proofread for such mistakes. After the incorrect base is incorporated, it will not remain hydrogen-bonded to the tautomeric base in the template once the latter returns, almost immediately, to its more stable form. The 3 — 5 exonuclease activity shows a strong preference for a frayed or non-hydrogen-bonded end and removes the misincorporated nucleotide before chain growth proceeds further. DNA polymerase III holoenzyme also has the potential to proofread by the same mechanism. 

The proofreading 3 —> 5 nuclease of the e subunit of DNA polymerase III holoen-zyme removes most of the misincorporated nucleotides that do not form a base pair with the template. The polymerase activity of the holoenzyme then has a second chance to incorporate the correct nucleotide. Additionally, DNA repair systems exist that can detect and repair a mismatched base pair resulting from a misincorporation during synthesis. 

Structural studies as well as sequence comparisons among polymerases strongly suggest the hypothesis that the phosphoryl transfer reaction of all polymerases is catalyzed by a two metal ion mechanism that was originally proposed by analogy to the well studied two metal mechanism in the 3 exonuclease reaction (14). It is perhaps of interest to note that such a mechanism, which involves only the properties of two correctly positioned divalent metal ions, could easily be used by an enzyme made entirely of RNA and thus could function in an all RNA world. The fidelity of DNA synthesis appears to arise from two sources. First, enforced Watson-Crick interactions at the polymerase active site increases the accuracy of the incorporation step (9,13). Second, there is a competitive editing at the 3 exonuclease active site that removes misincorporated nucleotide (3,5). When nucleotides are... 

It is now believed that a substantial proportion of the single nucleotide substitutions causing human genetic disease are due to misincorporation of bases during DNA replication. Which proofreading activity is critical in determining the accuracy of nuclear DNA replication and thus the base substitution mutation rate in human chromosomes ... 

To minimize the misincorporation of different nucleotides during PCR reaction, PCR amplification is performed using LA Taq polymerase (Takara) since this has relatively high fidelity, and the number of PCR cycles is kept to 20. Excess primers and dNTPs are treated with shrimp alkaline phosphatase and Exonuclease I. 

The dependence of overall misincorporation on the concentration of the next nucleotide to be incorporated provides a diagnostic test for the active participation of an editing mechanism. 

Even with its proofreading activity, E. coli DNA polymerase III still exhibits a measurable rate of nucleotide misincorporation (about one mistake per 1010 nucleotides incorporated). Mutants of E. coli DNA polymerase III can be isolated that have a lower than normal rate of misincorporation. Why might such mutants, which can be said to have hy-peraccurate DNA replication, be evolutionarily unfavorable ... 

Figure 2 Structure of DNA polymerases, (a) The structure of Pol I represents the general right-hand shape of all DNA polymerases (PDB ID IQSY). Subdomains and the 3 -5 exonuclease domain are indicated, (b) The chain topology in the palm domain of DNA polymerases indicates their evolutionary heritage. Left The A, B, and Y family have four antiparallel sheets supported by two a helices. The three aspartic residues that chelate the metal ions are indicated by dots. Right The C-famiiy and X-family DNA polymerases contain a set of paraiiei strands on which the acidic residues that chelate metal are oriented differently from the A-, B-, and Y-family DNA polymerases, (c) During misincorporation of an incorrect nucleotide, the DNA must leave the polymerase active site (left) and enter the 3 -5 exonuclease active site (right). This action requires the melting of three to four base pairs.

The low error rate of nucleotide misincorporation during DNA synthesis is due to... 

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